Autophagy mediates degradation of cytoplasmic material through the lysosomes, and inefficiencies in this vital homeostatic process result in devastating diseases. For example, neurodegenerative lysosomal storage disorders are characterized by deficiencies in lysosomal degradation and autophagy dysfunction. Inefficient autophagy also leads to accumulation of aggregated proteins and neurodegeneration in protein misfolding diseases such as Parkinson's disease. Thus, translational research strategies to activate autophagy could dramatically impact the development of therapeutics for a large range of neurodegenerative diseases. Interestingly, evidence of autophagy induction by nanomaterials has recently emerged. Unfortunately, most current synthetic nanoparticle systems are unable to cross the blood-brain barrier (BBB), precluding their use to treat the central nervous system. In addition, most nanomaterials disrupt lysosomal function, ultimately leading to block of autophagy flux and impaired clearance. To overcome these limitations, we propose to develop a platform of virus nanoparticles (VNP) that can induce the coordinated activation of autophagy and lysosomal biogenesis. Our VNP technology is based on the adeno-associated virus (AAV), and will particularly focus on AAV serotype 9 that was shown to cross the BBB. Evidence of the integrated and co-regulated roles of lysosomes and autophagosomes emerged from the recent discovery of a master regulator of autophagy and lysosome biogenesis, the transcription factor EB (TFEB). The long-term goal of this work is the development of a VNP-based platform technology to activate TFEB and enhance clearance of autophagic cargo by lysosomes. The objective of this proposal is to identify the design rules for generating VNPs that can activate TFEB and enhance clearance in vitro. The central hypothesis of this study, based on our pilot data, is that the TFEB-activating properties of VNPs depend on cellular uptake of the particle and not on the infectivity of the capsid since a defective VNP unable to productively infect cells can still activate TFEB. If successful, the proposed research will produce an enabling therapeutic platform for the treatment of neurodegenerative diseases characterized by accumulation of autophagic substrates. Specifically, we propose to define the design rules for building VNP-based activators of TFEB (aim 1), and we will test the performance of VNPs in vitro by evaluating clearance of i) lipofuscin in fibroblasts derived from patients with Neuronal Ceroid Lipofuscinosis and ii) aggregated -synuclein in neuroglioma cells (aim 2). The proposed research is significant because it will generate a therapeutic platform able to promote co-regulated activation of the lysosome-autophagy system for effective treatment of neurodegenerative diseases. This approach is innovative because it promises to overcome limitations of currently available synthetic nanomaterials that cannot cross the BBB. Results from this study will also significantly advance our knowledge of AAV biology, as no scientific information is currently available concerning the impact of AAV on the autophagy pathway.